1. Field of the Invention
The present invention relates to nucleic acid sequences derived from fungal genes which encode polypeptides having cell wall-degrading activity and isolated polypeptides having cell wall-degrading activity. The invention also relates to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant""s resistance to Fusarium and other pathogens.
2. Description of the Art
Wheat is one of the most important food crops for both domestic and export markets. The United States produces about 2.4 billion bushels of wheat per year with a value of over 7 billion dollars. Fusarium head blight or scab is a ftngal disease of wheat, barley, oats, rye, and wheatgrasses that affects both grain yield and quality. It occurs worldwide, particularly when temperatures and humidity favor the proliferation of the causal agent, Fusarium graminearum, at the time of heading. Head blight has caused losses in the billions of dollars to United States and Canadian growers and processors within this decade. Yield and grain quality losses of wheat due to Fusarium head blight approached one billion dollars in Minnesota, North Dakota, and South Dakota in 1993 and 200-400 million dollars across the region in subsequent years. Losses were in excess of 300 million dollars in Ohio, Michigan, Indiana, and Illinois in 1995 and 1996. Quality of grain is also compromised since infected grain is usually contaminated with a mycotoxin, vomitoxin or DON, produced by the fungus that is detrimental to humans and livestock. In addition, the disease has threatened barley production in the upper Midwest because brewers have imposed zero tolerance limits for vomitoxin in grain.
Cereal crops, including wheat, maize, barley, oats, and rye, are susceptible to infection by many types of fungi and by many species of the pathogenic fungus Fusarium. Fusarium graminearum (Schwabe) and Fusarium culmorum are the primary causal agents of a disease known as Fusarium head blight, head blight, or scab, of wheat, barley, oats and rye (reviewed in Bai and Shaner 1994; Parry et al. 1995). The life cycle of the pathogen alternates between two hosts, wheat and maize. The teliomorph (sexual stage) of F. graminearum, called Gibberella zeae, causes stalk rot, ear rot and seedling blight of maize. Head blight is a problem of wheat and barley worldwide, and has occurred in epidemic proportions in the United States since 1991. Thus far, no effective resistance genes for Fusarium head blight have been identified in wheat, barley or their sexually-compatible relatives. Due to this deficiency, and as a consequence of no-till agriculture and unfavorable climate patterns, the wheat and barley industries in 12 states have sustained direct losses totaling 2.6 billion dollars. Increased accumulation of corn and wheat stubble has resulted from no-till agriculture and contributes to the propagation of spores and conidia in the field. Moreover, periods of rainfall and warm temperatures at the time of anthesis (pollen shed) favor germination and growth of the pathogen. F. graminearum causes death of the floral organs (florets) that harbor the developing grain, giving the head a bleached and xe2x80x9cscabbyxe2x80x9d appearance, and resulting in moderate to severe reductions in grain yield. In addition, F. graminearum and other Fusarium species produce trichothecene mycotoxins, such as deoxynivalenol (DON), that exacerbate disease severity and pose a health threat to humans and livestock that ingest contaminated cereal products.
The marked susceptibility of the wheat head to Fusarium was noted as early as 1891 by Arthur (Parry et al. 1995). Pugh and coworkers (1933) reported that wheat heads inoculated with cultured conidia of F. roseum (culmorum) were most susceptible to infection during a 20-day window, from anthesis to the soft dough stage, depending upon the cultivar. Dehisced anthers and other degenerating tissue appeared to serve as foci for the proliferation of hyphae into the phloem and throughout the rest of the head. In histological studies, Pugh noted that hyphae usually invade intercellularly but can penetrate the thin walls of the inner parts of the floret. Although F. graminearum can become established on various parts of the flower, rapid spread of infection was correlated with the presence of extruded anthers both in laboratory and field studies (Andersen 1948, McKay and Loughnane 1945). Aqueous extracts of anthers were found to stimulate growth of hyphae in vitro (Strange and Smith 1971), whereas germination of macroconidia appeared to be unaffected (Strange and Smith 1978). Hyphal growth stimulation was almost entirely attributable to two quaternary ammonium compounds, glycinebetaine and its precursor choline (Strange et al. 1974). These compounds are present in other organs of the floret, but are most abundant in pollen (Pearce et al. 1976). Glycinebetaine, an osmoprotectant, accumulates over the normal course of pollen development during desiccation (reviewed in McCue and Hanson 1990). Along with choline, it is postulated to serve as a fortuitous source of carbon and nitrogen for F. graminearum and for other fungal pathogens. Whether fungal hyphae readily access these compounds at the pollen surface or must invade the pollen cytoplasm is not known at this time.
Identification of genes that confer resistance to Fusarium head blight is essential if wheat and barley are to remain in production in Minnesota, North Dakota, and other states. If Fusarium infection can be curtailed by even 10%, the economic impact is expected to be millions of dollars saved by producers, processors and, ultimately, consumers. Multigenic loci for resistance to scab have been identified in germplasms of wheat and other cereals, but at present, the degree of tolerance obtained in adapted cultivars through traditional breeding is inadequate for control of the pathogen. Since no-till agriculture offers clear benefits for soil and water conservation and since Fusarium is so abundant in wheat-growing regions, alternative sources for host resistance are needed.
There are no effective control measures for the disease. Resistance genes for Fusarium head blight have not been identified in wheat, barley or other sexually-compatible species, limiting the efforts of wheat breeders to develop resistant varieties. What are needed are ways to obtain crop varieties that are resistant to Fusarium head blight.
In many fungi, including species of Fusarium, glucan and chitin are principle components of the cell wall. The outer wall layer of Fusarium is comprised of polymers of glucose (1,3/1,6-xcex2-D-glucan) with xcex2-1,3 and xcex2-1,6 linkages. The basal-most inner layer consists primarily of chitin microfibrils, linear polymers of xcex2-1,4-N-acetylglucosamine that account for about one-third of the mass of the hyphal wall (Barbosa and Kemmelmeier 1993). Fusarium cell walls appear to be more refractory to the action of hydrolytic enzymes, possibly because of high levels of protein and chitin (Sivan and Chet 1989a), and clustering of acetylated glucosamine residues (Fukamizo et al. 1992).
Chitinases and glucanases are produced by naturally occurring bacteria, fungi and plants. In the fungi, these proteins have roles in the self-hydrolysis of cell wall chitin and glucan, respectively, and in the hydrolysis of cell wall components of other microorganisms (Srivastava et al. 1985; Sivan and Chet 1989b, Chxc3xa9rif and Benhamou 1990, Vxc3xa1zquez-Garciduexc3x1as et al. 1998). This latter feature has been applied to the discovery of anti-microbial biocontrol agents. Chitin is also found in the cuticles of insects and in nematode egg shells. For fungi that can utilize chitin as a carbon source, chitinase might have a role in fungal metabolism (Flach et al. 1992). Endochitinases cleave randomly between C1 and C4 linkages within the chitin polymer (Flach et al. 1992, Graham and Sticklen 1994), whereas exochitinases cleave sequentially between each linkage, releasing chitobiose from the terminus of the chitin polymer. N-acetyl-glucosaminidase also cleaves terminally, releasing N-acetylglucosamine (Flach et al. 1992). Likewise, endoglucanases randomly cleave xcex2-linkages within the glucan polymer, generating short oligosaccharides, whereas the exoglucanases cleave single glucose residues from the nonreducing end of the polymer (Vxc3xa1zquez-Garciduexc3x1as et al. 1998). Hydrolysis of the glucan layer of fungal cell walls is attributed to the combined action of both endo- and exo-glucanases.
The distinctive glucan and chitin composition of the fungal cell wall has led to extensive engineering of chitinases and glucanases as antifungal proteins. Chitinases and glucanases produced by plants have been widely characterized as pathogenesis-related (Pr) proteins involved in defense against pathogens and insect pests. Pr protein genes in plants are induced upon exposure to microbial pathogens and insect pests. Cell wall-degrading proteins have been grouped into classes on the basis of their biochemical and structural properties. Both endo- and exochitinases appear to be effective in the hydrolysis of fungal cell wall chitin. Class 1 chitinases have chitinolytic activity against bacterial cell walls and are known to bind chitin directly. By contrast, the class II chitinases do not act on bacterial cell walls and lack chitin binding activity; they are postulated to play a role in production of fungal elicitors that trigger the host defense response (Graham and Sticklen 1994, Fritig et al. 1998). In general, the basic chitinases and glucanases (class I) accumulate intracellularly in the vacuole, and the acidic isoforms (class II) are extracellular, with some exceptions (e.g., Wu et al. 1994; Graham and Sticklen 1994). Comelissen and coworkers (Sela-Buurlage et al. 1993) and others (Graham and Sticklen 1994) observed that the specific activities of class I chitinases and glucanases are higher than those of the class II enzymes. Class 1 chitinases are encoded by small gene families in most plants and in the fungi. The structure of various chitinase genes and proteins has been reviewed (Graham and Sticklen 1994).
Of the five major classes of xcex2-glucanases, only the xcex2-1,3-glucanases have been shown to exhibit antifungal activity (Simmons 1994). The xcex2-1,3-glucanases are members of small multigene families in plants (Payne et al. 1990, Xu et al. 1992, Beffa and Meins 1996, Simmons 1994), but are single-copy genes in Fusarium sporotrichioides (FIG. 27). Class I glucanases accumulate in the vacuoles, whereas Class II and III glucanases are acidic and extracellular (see Beffa and Meins 1996). Class I glucanases have been widely studied in the context of pathogen defense and stress responses in tobacco (Linthorst et al. 1990; Linthorst 1991; Payne et al. 1990), barley (Jutidamrongphan et al. 1991; Xu et al. 1992; Malehom et al. 1993), wheat (Jutidamrongphan et al. 1991; Cruz-Ortega et al. 1997), and other species (Krishnaveni et al. 1999a, reviewed in Simmons 1994). Chitinases and glucanases act preferentially on the tips of growing fungal hypha (for instance, Broekaert et al. 1988, Collinge et al. 1993). Both chitinases and xcex2-1,3-glucanases are differentially expressed during plant development (Lotan et al. 1989) as well as in response to pathogen attack. Glucanases appear to have roles in cell division and flower development (Beffa and Meins 1996) and the stress response (Simmons 1994) in plants. The role of chitinases in plant development is not as well-characterized; however, they have been implicated in embryogenesis and cell division (for review, see Collinge et al. 1993)
Some of the chitinases and xcex21,3-glucanases produced by naturally occurring bacteria and fungi have anti-Fusarium properties (Mitchell and Alexander 1961; Michael and Nelson 1972, Chxc3xa9rif and Benhamou 1990). Glucanases and chitinases from plants can degrade isolated cell walls of Fusarium solani (Mauch et al. 1988). Chitinases from tobacco were inhibitory to the growth of F. oxysporum (Yun et al. 1996) and F. solani (Sela-Buurlage et al. 1993) in culture. Krishnaveni et al. (1999b) have described three chitinases from sorghum seeds that inhibit the growth of F. moniliforme. The synergistic action of chitinases and glucanases against fungal pathogens is widely reported (reviewed in Graham and Sticklen 1994, Van Loon 1997). For instance, Mauch et al. (1988) observed that a chitinase and a xcex21,3-glucanase from pea wee active against a wide range of fungi. Melchers et al. (1994) reported the combined action of a Class V endochitinase plus a Class I xcex21,3-glucanase, both from tobacco, synergistically inhibited the growth of F. solani. Expression of a tobacco acidic chitinase with a tobacco xcex21,3-glucanase conferred resistance to F. oxysporum in tomato, whereas each protein had much less effect when expressed singly (Jongedijk et al. 1995). Likewise, the fungal pathogen Cercospora nicotiana was curtailed on tobacco expressing both a rice basic chitinase and an alfalfa acidic xcex2-1,3-glucanase (Zhu et al. 1994). The synergistic action of a barley Class II chitinase and a barley Class II b-1,3-glucanase conferred protection to tobacco against Rhizoctonia solani (Jach et al. 1995). This chitinase in combination with a barley ribosome inactivating protein also inhibited R. solani infection.
Although fungi and many, if not all, plants express glucanases and chitinases, not all of the enzymes are equally effective against all types of microorganisms (Graham and Sticklen 1994, and references therein). For example, Broekaert and co-workers (1988) observed that chitinases from thorn-apple, tobacco and wheat had antifungal activity against Trichoderma harzianum and Phycomyces blakesleeanus but not against Botrytis cinerea. Mauch et al. (1988) reported the differential action of a chitinase and a glucanase from pea on Trichoderma viride and F. solani, respectively. In combination, the enzymes were active against a wide range of other fungi. A tobacco chitinase with activity against Fusarium and Trichoderma were inactive against Aspergillus flavus, Phytophthora parasitica and other pathogens (Yun et al. (1996). The differential activities of the chitinases are attributed to inherent properties of the enzymes (Sela-Buurlage et al. 1993, Brunner et al. 1998), to differences in cell wall architecture (Sivan and Chet 1989a, Van Loon 1997) among the fungi, or to other factors.
Additional types of proteins have been found to have anti-Fusarium activity in vitro or in planta. Boyapati et al. (1994) reported a cysteine protease inhibitor from pearl millet that inhibited the growth of Fusarium moniliforme in culture. A cysteine-rich polypeptide from Impatiens balsamina seeds was active against F. culmorum (Tailor et al. 1997). Cecropin A, a polypeptide from the Cecropia moth, was a potent inhibitor of both F. moniliforme and F. oxysporum (deLucca et al. 1997, Cavallarin et al. 1998). Antifungal proteins from seeds of sorghum had activity against F. moniliforme (Seetharaman et al. 1997), and two wheat seed proteins of the PR4 family of pathogenesis-related proteins inhibited hyphal growth of F. culmorum and F. graminearum (Caruso et al. 1996). Hu and Reddy (1997) isolated a thaumatin-like protein from Arabidopsis thaliana with activity against F. oxysporum. Non-specific lipid transfer proteins from barley and maize leaves were inhibitory to F. solani (Molina et al., 1993). A combination of a wheat purothionin and a 2S albumin from radish or oilseed rape was effective against the growth of F. culmorum in vitro (Terras et al. 1993).
A majority of antifungal genes that have been examined both in vitro and in planta are of plant origin. To our knowledge, there are two examples of genes from fungi that exhibit antifungal activity. Endochitinases from the parasitic fungus Trichoderma harzianum conferred activity against Alternaria alternata and B. cinerea in transgenic tobacco, and against A. solani and Rhizoctonia solani in transgenic potato (Lorito et al. 1998). Terakawa et al. (1997) observed protection of transgenic tobacco against Sclerotinia sclerotiorum and B. cinerea, using a chitinase gene from the fungus Rhizopus oligosporus. A chitinase from the bacteria Serratia marcescens showed antifungal activity when expressed in tobacco (Suslow et al. 1988).
The present invention is directed to nucleic acid sequences derived from Fusarium fungal genes which encode polypeptides having cell wall-degrading activity as well as isolated polypeptides having cell wall-degrading activity. The invention is also directed to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences and methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant""s resistance to Fusarium and other pathogens.
More particularly, the invention provides isolated nucleic acid molecules that encode polypeptides having cell wall-degrading activity comprising glucanase, endochitinase or exochitinase activity. Genomic sequences encoding glucanase and exochitinase and cDNA sequences encoding glucanase, endochitinase and exochitinase are specifically exemplified herein. Included within the scope of this invention are nucleic acid sequences encoding a polypeptide having the glucanase, endochitinase or exochitinase polypeptide sequences exemplified below and nucleic acid molecules encoding polypeptides having glucanase, endochitinase or exochitinase activity.
Nucleic acid sequences which hybridize specifically to an enzyme coding sequence or its complement under medium or high stringency conditions and which encode a polypeptide having glucanase, endochitinase or exochitinase activity are also encompassed by the present invention.
Nucleic acid sequences having at least 70% sequence identity with the exemplified glucanase sequences as described in detail, below, and which encode a polypeptide having glucanase activity are also encompassed by the present invention. Nucleic acid sequences encoding a polypeptide having at least 80% sequence identity with the exemplified glucanase polypeptide sequences as described in detail, below, and which encode a polypeptide having glucanase activity are also encompassed by the present invention.
Nucleic acid sequences having at least 75% sequence identity with the exemplified endochitinase or exochitinase sequences as described in detail, below, and which encode a polypeptide having endochitinase or exochitinase activity are also encompassed by the present invention. Nucleic acid sequences encoding a polypeptide having at least 85% sequence identity with the exemplified endochitinase or exochitinase polypeptide sequences as described in detail, below, and which encode a polypeptide having endochitinase or exochitinase activity are also encompassed by the present invention.
The present invention is also directed to isolated polypeptides having glucanase, endochitinase or exochitinase activity. A polypeptide having an amino acid sequence which has at least 80% sequence identity with exemplified glucanase polypeptides as described in detail, below, is encompassed by the invention. A polypeptide having an amino acid sequence which has at least 85% sequence identity with exemplified endochitinase or exochitinase polypeptides as described in detail, below, is also encompassed by the invention. Polypeptides encoded by a nucleic acid sequence which hybridizes under medium or high stringency conditions with exemplified nucleic acid sequences as discussed in detail, below, are also encompassed by the invention. Variants of the polypeptides are encompassed by the invention as well as fragments having glucanase, endochitinase or exochitinase activity.
The invention is also directed to methods of producing and using the polypeptides of the invention.
A further aspect of the invention is the provision of recombinant nucleic acid molecules containing the sequences encoding polypeptides having the fungal cell wall-degrading activity, including glucanase, endochitinase or exochitinase activity. Such molecules include, for example, recombinant vectors, such as cloning, expression or transformation vectors, which contain a DNA sequence encoding a glucanase, endochitinase or exochitinase.
Another aspect of the invention is the provision of cells which are transformed by the above vectors or DNA sequences.
A particular use of the invention is the provision of cells transformed with one or more nucleic acid sequences of the invention. A more particular use of the invention is the provision of plants, plant seeds or plant cells transformed with one or more nucleic acid sequences encoding a polypeptide having glucanase, endochitinase or exochitinase coding activity to provide plants having resistance to plant pathogens, including fungi, particularly, Fusarium species or to provide plants having enhanced resistance to plant pathogens.
A further aspect of the invention is the provision of oligonucleotide probes capable of detecting a glucanase, endochitinase or exochitinase gene or functional equivalents thereof in fungi of the genus Fusarium and the use of the probes to isolate nucleic acid sequences encoding a glucanase, endochitinase or exochitinase gene or functional equivalent thereof. The nucleic acid sequences which specifically hybridize to the probes and which encode a functional glucanase, endochitinase or exochitinase are encompassed by the present invention.
Using the nucleic acid sequences of the invention facilitates the isolation of homologous genes from fungi to obtain genes which protect host cells, including fungi, bacteria, and plants against related fungal pathogens.
The invention also includes the application of transgene constructs in combination with each other, that is, glucanase and endochitinase; glucanase and exochitinase; endochitinase and exochitinase; and glucanase, endochitinase and exochitinase. Also included are transgenic monocot or dicot lines, plant cells, and progeny obtained by sexual or asexual propagation, that carry any combination of the transgene constructs.
In accordance with this discovery, it is an object of the invention to provide nucleic acid sequences encoding fungal cell wall-degrading enzymes selected from the group consisting of glucanase, exochitinase, and endochitinase; isolated polypeptides having glucanase, endochitinase or exochitinase activity; recombinant nucleic acid molecules including expression vectors encoding polypeptides having cell wall-degrading activity; and cells harboring the recombinant nucleic acid molecules or expression vectors.
It is also an object of the invention to provide transformation vectors comprising a cell wall-degrading recombinant molecule, which vectors are effective for stably introducing the recombinant molecule into a plant.
It is also an object of the invention to provide methods of producing and using polypeptides having glucanase, endochitinase or exochitinase activity.
It is another object of the invention to provide transgenic plants having bacterial or fungal resistance, wherein the resistance is a result of expression of a recombinant nucleic acid molecule of the invention.
A further object of the invention is to provide fungal genes which generate cell wall-degrading enzymes, including proteins having the capability of degrading the glucan and chitin cell wall components of F. venenatum and other Fusarium species, including F. graminearum and F. culmorum, the principle causal agents of head blight (scab) in the U.S.
Another object of the invention is expression of the cell wall-degrading enzymes in transgenic monocots, including wheat, barley or oats, to confer partial or complete resistance to Fusarium species and/or to other fungal pathogens of wheat and other cereal crops. Such transgenic lines will be useful genetic stocks for generating improved crops.
A still further object of the invention is the provision of novel wheat germplasms that express genes designed to limit the spread of the pathogenic fungus Fusarium and indirectly to curtail the accumulation of DON in infected heads.